Since the food-borne bacteria are primarily transmitted through foods such as meat, dairy products, drinking water, agricultural foods, etc., a method by which the presence or absence of the food-borne bacteria in samples such as food can be confirmed quickly and economically is required. Conventional methods for detecting food-borne bacteria are culturing the sample in a selective medium to separate the bacteria which are estimated as the food-borne bacteria, and then confirming them by biochemical or immunological methods. However, in the immunological method using an antibody, it is possible to detect bacteria with high accuracy, but it requires a large number of samples, and for the production of antibody required for each diagnosis, the protein purification of the relevant bacteria, production, or manufacture of peptide is essential, and high cost for producing the antibody is needed. Furthermore, in view of the nature of the protein, there are many difficulties in use and storage of it and it can only detect one type or a limited kind of bacteria at one time, and a longer time is consumed in culturing bacteria. To improve these drawbacks, the detection kits of the various bacteria using the PCR method have begun to be researched and developed. Detection kits using PCR method have been increased in light of the demands in various fields because of a high accuracy, simplicity, and rapidity.
In particular, the method of real-time PCR being recently often used is that there is an increased observation in the PCR amplification product in real time for each cycle of the PCR, and that detecting and quantitatively analyzing a fluorescent substance that reacts with PCR amplification products. This method has advantages, compared to that the existing PCR methods in which the gel was stained after finishing the final step to make electrophoresis in order to verify the PCR amplification product. The advantages include: no additional work for electrophoresis is needed, precision and sensitivity are excellent, it has high reproducible rate, and the automation is possible, the result can be digitized, it is rapid and simple, a biological safety according to detrimental problems such as UV irradiation and pollution by dyes such as EtBr (Ethidium Bromide) is excellent, and it is possible to automatically confirm the presence or absence of amplification of specific genes. Thus, rather than a qualitative result such as PCR or antigen/antibody via the method of real-time PCR, it is possible to confirm the quantitative results with high specificity. Further, since it is a probe labeled with a fluorescent label factor, it can confirm the results of a sample even with an amount smaller than the amount to be used for a DNA chip or antigen/antibody reaction. Therefore, in order to rapidly and accurately diagnose infection cause by food-borne bacteria in food, a need for the development of detection methods and detection kits of food-borne bacteria using real-time PCR method is in demand.
Real-time PCR (real-time Polymerase Chain Reaction) has been recently used a lot in the execution of a nucleic acid amplification reaction because of the advantage that a nucleic acid amplification product can be confirmed in real time during the reaction cycle without running the electrophoresis on gel. In general, an apparatus for carrying out the real-time PCR includes a thermal cycler with one or more heat blocks to perform a nucleic acid amplification reaction and a signal detector for measuring the signal generated from the nucleic acid amplification product in real time. Such signal detectors can be embodied as: a photo detector for detecting a fluorescence signal generated from the nucleic acid amplification products, an electronic signal detector for detecting an electric signal generated through a specific binding of the nucleic acid amplification product and the mediator interconnecting with it, and the like.
Meanwhile, in a recent medical field, effective diagnosis and treatment methods for implementing a personalized medicine (tailor-made medicines) have been actively developing, and in order to substantially achieve a personalized medicine, there is a need for rapid and accurate diagnosis and treatment for a number of objects. In this case, it could be said that in the diagnosis and treatment, the nucleic acid amplification step is the pre-process which is mostly based, and the real-time PCR which is an example for performing this is the pre-step in the realization of personalized medicine. However, since the real-time PCR has the assumption of a complex execution process, it takes considerable time to complete the step, and the devices for recognizing this are mostly expensive and large, and thus, there has been a failure to realize the potential of personalized medicine. Recently, many attempts have been made to solve said problems.
In this regard, Korean Patent Publication No. 10-2004-0048754 (Temperature-controllable, real-time fluorescence detection apparatus) provides a portable compact fluorescence detection device wherein various wavelengths of fluorescence are searched rapidly within a few seconds even at a low concentration of the sample sensitively, wherein the enzyme reactions can be searched and analyzed in real time at an more economical price. Specifically, the said fluorescence-searching apparatus is the device analyzing the sample by searching fluorescence emitted from a biological sample after irradiating a light source to said sample, characterized by comprising a LED array disposed so that a plurality of LEDs sequentially emit the light; a well chamber block having a plurality of wells to insert the sample vessel; a multi-channel PMT to detect fluorescence emitted from said sample by each LED light-emitting of said LED array; and a plurality of optical fibers to transmit fluorescence emitted from said each sample to said multi-channel PMT, in a fluorescence-searching apparatus comprising a sample vessel, light source locating so as to irradiate the sample vessel, fluorescence-transmitting device, wavelength selection device, and controlling unit.
Also, Korean Patent Registration No. 10-0794703 (Real-time monitoring apparatus of biochemical reaction) provides an apparatus which can compare and analyze the reaction degree of various samples, by minimizing light detection sensitivity deviation upon the reaction in a reaction tube plate. Specifically, the previous real-time monitoring apparatus comprises a temperature-regulating block system consisting of the heat transfer block for transferring heat to the reaction tube and the thermoelectric element being a heat source capable of supplying heat to the reaction tube; the irradiation source unit consisting of a lamp and a optical waveguide for irradiating uniform light to the sample in the reaction tube; and an optical system composed of a reflecting mirror for changing an optical path, and a light receiving unit for receiving the fluorescence generated from the sample of the reaction tube by the light irradiated by the irradiation source unit.
Also, Korean Patent Registration No. 10-1089045 (Real-time monitoring device of the nucleic acid amplification reaction product) whose purpose is to monitor the generation of a reaction product produced during the reaction while performing a nucleic acid amplification reaction such as the polymerase chain reaction for a large number of samples in small amounts, and provide a real-time monitoring device comprising a polarizer, polarization beam splitter, polarization converter, and the like.
Further, Korean Patent Publication No. 10-2008-0103548 (Real-time detection apparatus of nucleic acid amplification products) provides a real-time detection system of the nucleic acid amplification products that can determine the strength [DNA] real of a plurality of wells, without using a second fluorescence signal used for correction, in order that error factors on the device can be eliminated or reduced, by applying a temperature cycle to a plurality of wells, detecting fluorescence strength from a nucleic acid amplification product in each well in real time, and further fluorescence measurement values obtained from the well, [DNA] raw, and fluorescence measurement values obtained from the periphery of the connection wall near the well, [DNA] bg, and subtracting the fluorescence measurement value [DNA] bg from the fluorescence measurement value [DNA] raw.
Also, Korean Patent Registration No. 10-0794699 (real-time monitoring device of the nucleic acid amplification reaction product) provides a real-time monitoring device of the nucleic acid amplification reaction product, characterized by comprising a transparent sealing cover to cover reaction vessel and the reaction vessel having many wells to receive many samples for monitoring the generation of a reaction product produced during the reaction in real time while performing a nucleic acid amplification reaction such as the polymerase chain reaction, of a large number of samples in small amounts; fluorescence element consisting of selective transmitting filter located in front of excitation light source, the line polarizer for line-polarizing the light passed through the filter; light-receiving element consisting of line-polarizer located in the direction perpendicular to the line polarizer of the light-emitting element, light-collecting lens for collecting light passed through the line polarizer, selective transmitting filter transmitting the light passed the light-collecting lens, and fluorescent-sensing element.
However, since the above-mentioned prior art utilize a large number of measurement modules consisting of complex and sophisticated fluorescence signals in order to measure a number of the nucleic acid amplification products at the same time, the large size of the device and high cost are still problematic. Further, although said prior arts' purposes are measure a large number of small amounts of samples simultaneously, they do not disclose any method for solving the phenomenon in which the signal sensitivity is greatly reduced by a bubble which in the nucleic acid amplification process is generated by heating in a small amount of the sample solution contained in the small reaction vessel.
Therefore, a real-time PCR mounting apparatus is still needed that is capable of making real-time monitoring of nucleic acid amplification product at a low cost more quickly in order to ensure the reliability of the measured values along with the measuring of a large number of small amounts of nucleic acid amplification products at the same time, this is also applicable in regards to the detection device which can simultaneously, quickly, and accurately detect a plurality of food-borne bacteria, and the detection method of food-poisoning bacteria by using this.